Abstract: To investigate the codon usage pattern and major influencing factors in the chloroplast genome of Parnassia amoena, the complete chloroplast genome of P. amoena was used as the research object. Through screening and removing duplicate genes, a total of 44 coding DNA sequences (CDS) were ultimately obtained. These sequences were analyzed using Codon W 1.4.2 and CUSP programs, including assessments of codon base composition, neutral plot analysis, ENC-plot analysis, PR2-plot analysis, and optimal codon identification. The results showed that the GC content at the three positions of codons in the chloroplast genome of P. amoena was GC₁>GC₂>GC₃, indicating a preference for A/T (U) at the end of codons. The average effective number of codons (ENC) in the genome was 48.05, indicating a relatively weak codon usage bias in P. amoena. In the correlation analysis, a highly significant positive correlation was observed between ENC and GC₃, suggesting that the nucleotide composition at the third codon position influences codon usage bias. The neutral plot results showed the codon usage bias is influenced by 48.15% mutation pressure and 51.85% selection pressure. Combined with ENC-plot and PR2-plot analyses, it further indicates that both base mutation and natural selection jointly influence the codon usage pattern in the chloroplast genome of this species, with natural selection playing a dominant role. Furthermore, optimal codon analysis identified 19 optimal codons (including UUA, AUU, and GUA) in the P. amoena chloroplast genome, most of which terminate with U, while the remainder end with A. The results of this study will provide theoretical references for the research on molecular evolution mechanism and chloroplast genetic engineering of P. amoena, and to lay a foundation for the study of systematic evolution of Parnassia.